JP4409287B2 - Assays identifying HIVRT dimerization inhibitors - Google Patents

Abstract

A method for measuring heterodimerization of HIV RT, which comprises the steps of:a) providing a first solution comprising p66 subunit homodimers in the presence of a dissociation agent; b) contacting the first solution with p51 RT subunits and incubating in the presence of a reassociation buffer to allow association of a complex of p66/p51 RT subunits, wherein one of the subunits comprises an affinity tag and the other of the subunits comprises a detectable label; c) contacting the incubate of step b) with an affinity medium under conditions that enable the p66/p51 complex to bind to the affinity medium; and d) determining the amount of complex formed by measuring the level of detectable label bound to the affinity medium (or by measuring the reconstituted RT polymerase activity). Steps a) to d) can be carried out in the presence or absence of a test compound followed by e) comparing the test compound sample to a control sample lacking the compound, whereby modulated p66/p51 complex formation in the test compound sample is indicative of the ability of the compound to modulate, inhibit or enhance heterodimerization. The method can be used to screen for inhibitors of HIV RT dimerization.

Description

Detailed Description of the Invention

TECHNICAL FIELD The present invention relates to a novel assay for measuring the dimerization process of HIV-1 reverse transcriptase (RT). In particular, the present invention relates to a novel method suitable for application to high-throughput screening for drugs that inhibit the dimer formation process.

BACKGROUND ART Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) plays a major role in HIV replication by converting single-stranded genomic RNA into double-stranded proviral DNA, and aids It has become one of the major targets in therapeutic development. Most of the RT inhibitors reported so far have targeted RT polymerase activity, whether nucleoside analogues or non-nucleoside inhibitors, but have some limitations, including the emergence of toxic and resistant strains .
The biologically relevant and active form of HIV RT found in infectious virions is a heterodimer containing two polypeptides (p66 and p51). p51 is derived from p66 by proteolytic cleavage of the C-terminal domain of p66 during viral maturation. The two subunits of the 66 kDa and 51 kDa forms are present in a 1: 1 ratio. This heterodimer RT is generated in a two-step dimer formation process. The kinetics involves the process of two subunits rapidly assembling to form immature dimers, followed by a slow structural change that produces a fully active form (p66 + p51-> immature p66 / p51-> active p66 / p51 (Divita et al. 1995, J. Mol. Biol. 245: 508-521, 12, 13)).
The DNA polymerase and ribonuclease H activities of HIV-1 RT depend on the dimer structure of this enzyme (Restle et al. 1990, J. Biol. Chem. 265: 8986-8988; Restle et al. 1992, FEBS Letters 300: 97- 100). Since dimer formation of these subunits is required for enzyme activity, inhibition of HIV-1RT dimer formation would be a suitable target for the development of anti-HIV compounds. Case: Synthetic peptides derived from the thumb domain of the p51 subunit of RT inhibit the “maturation” process (Morris et al. 1999, Biochemistry 38: 15097-15103). Compounds that impair the production and / or stability of RT dimers will therefore be a new class of antiviral compounds.

Several publications have disclosed association-dissociation assays aimed at measuring the kinetics of the p51-p66 dimer formation process (Cabodevilla et al. 2001, Eur. J. Biochem. 2681: 1163-1172; Morris et al 1999, J. Biol. Chem. 274 (35): 24941-24946; Divita et al. 1995, Biochemistry 34: 16337-16346; Divita et al. 1994, J. Biol. Chem. 269 (18): 13080- 13083; Divita et al. 1993, FEBS Letters 324 (2): 153-158; Becerra et al. 1991, Biochemistry 30: 11707-11719). All of the above assays measure dimer formation by either: size exclusion HPLC; measurement of RNA-dependent DNA polymerase activity of samples; immunoprecipitation; or monitoring of intrinsic fluorescence emission of proteins. None of the above documents disclose binding assays suitable for the HTS format.
Tachedjian et al. (2000, PNAS 97 (12): 6334-6339) disclosed a yeast two-hybrid system to investigate the association-dissociation process of RT dimer formation. However, the system is not readily adaptable to high throughput assays that evaluate large amounts of potential inhibitors.
Howard et al. (1991, J. Biol. Chem., 266 (34): 23003-23009) proposed an assay system that monitors therapeutic agents that act by inhibiting dimer formation. This document also does not disclose a binding assay that can be used for high-throughput screening suitable for identifying potential inhibitors of heterodimer formation.

SUMMARY OF THE INVENTION The present invention provides a high throughput assay suitable for determining a large number of compounds for activity against the dimerization of HIV RT. The general principle of the assay of the present invention is that p66 / p66 homodimer (appropriately labeled with a detectable moiety and / or affinity) in the presence of a limiting concentration of dissociator (ie denaturant (eg urea)). Tag is attached), the p51RT subunit is contacted, and the mixture is incubated in the presence of a denaturant-free (or denatured agent-reduced) buffer to incubate the RT subunit. Reassociating followed by affinity capture of any reconstituted p51 / p66RT heterodimer. After washing to remove unbound material, the amount of affinity binding material is determined by measuring the level of detectable component (alternatively by measuring the reconstituted RT polymerase activity (in this case the p66 sub- The unit does not necessarily have to be labeled))) evaluated, which is proportional to the amount of affinity-bound labeled p66 / p51RT heterodimer. The assay is performed in the presence or absence of the test compound, whereby changes in the association of the p66 monomer subunit and the p51 subunit in the presence of the test compound relative to the control indicate that the test compound is an RT dimer. Indicates a regulator of formation.

In a first aspect of the invention, a method for measuring heterodimer formation of HIV RT is provided. The method includes the following steps:
a) providing a first solution comprising a p66 subunit homodimer in the presence of a dissociator;
b) contacting the first solution with the p51RT subunit and incubating in the presence of a reassociation buffer to allow association of the p66 / p51RT subunit complex, wherein one of the subunits is an affinity tag The other of the subunits includes a detectable label;
c) contacting the incubate of step b) with the affinity medium under conditions that allow the p66 / p51 complex to bind to the affinity medium; and d) determining the level of detectable label bound to the affinity medium. By measuring (or by measuring the reconstituted RT polymerase activity), the amount of complex produced is determined.
In a second aspect of the invention, there is provided a method for identifying a compound capable of modulating HIV RT heterodimer formation comprising:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) a test compound sample is compared with a control sample lacking the test compound, whereby the p66 of the test compound sample is compared. Modulation of / p51 complex formation indicates the ability of the compound to modulate heterodimer formation.

In a third aspect of the invention, a method is provided for identifying compounds capable of interfering with HIV RT heterodimer formation, including:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) a test compound sample is compared with a control sample lacking the test compound, whereby the p66 of the test compound sample is compared. A decrease in / p51 complex formation indicates the ability of the compound to inhibit heterodimer formation.
In a fourth aspect of the invention, there is provided a method for identifying a compound capable of enhancing HIV RT heterodimer formation comprising:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) comparing a test compound sample with a control sample lacking said test compound, thereby Increased p66 / p51 complex formation indicates the ability of the compound to enhance heterodimer formation.
In a fifth aspect of the invention, there is provided a method for measuring HIV RT homodimer formation comprising the following steps:
a) providing a first solution comprising a first p66 subunit homodimer in the presence of a dissociator;
b) contacting the first solution with a second p66 subunit homodimer in the presence of the dissociator and further in the presence of a reassociation buffer to allow association of the p66 / p66RT subunit complex. Wherein one of the subunits comprises an affinity tag and the other of the subunits comprises a detectable label;
c) contacting the incubate of step b) with the affinity medium under conditions that allow the p66 / p66 complex to bind to the affinity medium; and d) the level of detectable label bound to the affinity medium. Is determined (or by measuring the reconstituted RT polymerase activity) to determine the amount of complex produced.
In a sixth aspect of the invention, there is provided a method for identifying a compound capable of modulating HIV RT homodimer formation comprising:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) a test compound sample is compared with a control sample lacking the test compound, whereby the p66 of the test compound sample is compared. Regulation of / p66 complex formation indicates the ability of the compound to regulate homodimer formation.

In a seventh aspect of the invention, there is provided a method for identifying a compound capable of interfering with HIV RT homodimer formation comprising:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) comparing a test compound sample with a control sample lacking said test compound, thereby A decrease in p66 / p66 complex formation indicates the ability of the compound to inhibit homodimer formation.
In an eighth aspect of the invention, there is provided a method for identifying a compound capable of enhancing HIV RT homodimer formation comprising:
Steps a) to d) above are carried out in the presence or absence of a test compound; further e) a test compound sample is compared with a control sample lacking the test compound, whereby the p66 of the test compound sample is compared. Increased / p66 complex formation indicates the ability of the compound to enhance homodimer formation.
In a ninth aspect of the invention, a kit is provided for testing compounds with the potential to modulate HIV RT heterodimer formation. The kit includes a plurality of affinity-tagged p66 subunit homodimers, a plurality of labeled p51RT subunits, a dissociator, a reassociation buffer, an affinity medium, and a subunit to identify test compounds that bind to the transcriptase. Includes instructions for the embodiment to be used.
In a tenth aspect of the invention, a kit is provided for testing compounds with the potential to modulate HIV RT homodimer formation. The kit uses a plurality of affinity-tagged p66 subunit homodimers, a plurality of labeled p66RT subunits, a dissociator, a reassociation buffer, an affinity medium, and a subunit to identify test compounds that bind to the transcriptase. Includes instructions for the embodiment to be used.
Other objects, advantages and features of the present invention will become apparent from the following description of preferred embodiments with reference to the accompanying drawings, which are exemplary and should not be construed as limiting the scope of the invention. It will become clear by reading the technical description.

DESCRIPTION OF PREFERRED EMBODIMENTS
Unless defined or otherwise specified, all technical and academic terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
As used herein, the term “label”, “detectable label” or “detectable marker” can be linked to p66 or p51 to detect, measure and quantify subunits directly or indirectly. It refers to any group that allows the recognition of subunits. Examples of such “labels” include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes and affinity tags (eg biotin). Such a label is attached to p66 or p51 by well known methods. In the present invention, one label or a plurality of labels can be introduced at any position of p66 or p51. For example, the label can be present either at the C-terminus or N-terminus of the protein or within the main chain thereof.

  As used herein, the term “affinity label” or “affinity tag” refers to a label, which is specifically captured by a complementary affinity ligand. Examples of affinity tag / affinity ligand pairs include maltose binding protein (MBP) / maltose; glutathione S transferase (GST) / glutathione; streptavidin tag / streptavidin or neutravidin; or histidine (His) / metal. However, it is not limited to these. The metal used as the affinity ligand can be selected from the group consisting of cobalt, zinc, copper, iron and nickel (Wong et al. (1991), Separation and Purification Methods, 20 (1): 49-106). The affinity tag of the present invention can be introduced at any position of p66 or p51. For example, the tag can be present either at the C-terminus or N-terminus of the protein or within its main chain, but is preferably present at the N-terminus of the protein. Preferably the selected metal is nickel. The affinity ligand can be set on a solid phase, such as a bead, a well of a microplate, or a column to facilitate separation by affinity chromatography. The affinity tag can be introduced into p66 or p51 by gene recombination using a known method.

The terms “monomer” and “monomer p66” or “monomer subunit” refer to the single subunit of dimer p66. The monomer subunit may be an exact replica of a naturally occurring monomer subunit, or may be a biologically active analog or a biologically inactive analog (negative dominant). Good.
As used herein, the term “subunit” when referring to either p66 or p51 refers to one or two components of homodimeric RT (p66 / p66) or heterodimeric RT (p66 / p51). means.
As used herein, the term “homodimer” refers to a dimeric molecule (eg, p66 / p66) in which both subunits are the same.
As used herein, the term “homodimer formation” refers to the process by which the same subunit (ie, p66) forms a dimer.
As used herein, the term “heterodimer” refers to a dimeric molecule in which the two constituent subunits are different (eg, p66 and p66).
As used herein, the term “heterodimer formation” refers to the process by which two different subunits (ie, p66 and p51) form a dimer.
As used herein, the term “dissociator” refers to a substance that can dissociate a multi-component complex into individual subunits.
As used herein, the term “reassociation buffer” refers to a buffer that facilitates the association of individual subunits into a multi-component complex.
As used herein, the term “free of denaturant” refers to a state in which the dissociator is no longer present in an amount sufficient to prevent the binding of most subunits.
The term “affinity medium” as used herein is a solid support, such as a well, bead or column of a microtiter plate, coated with an affinity ligand suitable for capture of an affinity tag.

Preferred Embodiments A first preferred feature of the first embodiment provides a method for measuring heterodimer formation of HIV RT. In said method, preferably the first solution of step a) comprises a buffer selected from Tris-HCl, HEPES or Bis-Tris. More preferably, the buffer is Tris-HCl at a concentration of 0 mM to 50 mM. Most preferably, the buffer is Tris-HCl at a concentration of 25 mM.
Preferably, the first solution of step a) comprises a salt selected from Na ₂ SO ₄ or MgCl ₂ . More preferably, the salt is MgCl ₂ at a concentration of 0 mM to 100 mM.
In a preferred feature of the first embodiment, the dissociator in step a) is selected from urea, guanidine hydrochloride or acetonitrile. More preferably, the dissociator is urea at a concentration of 1 to 3.5M.
Preferably, the reassociation buffer of step b) is the same as the buffer used for dissociation of the p66 / p66 subunit, but preferably does not contain a denaturing agent. More preferably, the reassociation buffer is present in an excess amount relative to the denaturant buffer, and the dissociator can be diluted sufficiently to allow reassociation of the p66 and p51 subunits. Most preferably, the reassociation buffer is added in a 10-fold excess over the dissociation buffer.
Preferably, the reassociation buffer of step b) further comprises a salt selected from Na ₂ SO ₄ or MgCl ₂ . More preferably, the salt is Na ₂ SO ₄ at a concentration of 0 mM to 100 mM. Most preferably, the salt is Na ₂ SO ₄ at a concentration of 50 mM.

Preferably, p51 of step b) has an affinity tag attached or has a detectable label. More preferably, an affinity tag is added to p51 of step b).
Preferably, p66 in step b) has a detectable label or has an affinity tag attached. More preferably, p66 in step b) has a detectable label.
Preferably, the affinity tag of step b) is selected from the group consisting of a hexahistidine tag, FLAG, T7, HA, GST or biotin. More preferably, the affinity tag is hexahistidine.
In another preferred feature of the first embodiment, in step b), the detectable label is selected from the group consisting of: a fluorescent label (eg fluorescein, Oregon green, rhodamine, Texas red, phycoerythrin or Eu ^{3 +} ), A radioactive atom (eg ³ H or ¹²⁵ I), a chemiluminescent label (eg luciferin), a detectable antibody specific to p66 or a tag linked to p66 (preferably the tag of p66 is hexa A histidine tag, FLAG, T7, HA, GST or biotin), and an enzyme marker (eg, β-galactosidase or horseradish peroxidase). More preferably, the label is a fluorescent label selected from the group consisting of fluorescein, Oregon green, rhodamine, Texas red, phycoerythrin and Eu ³⁺ . Most preferably, the fluorescent label is Eu ³⁺ .
Preferably, the detectable label of step b) is added to a cysteine residue introduced by genetic recombination instead of the N-terminal proline residue of p66.
Said detectable label is measured by suitable means well known to those skilled in the art, such as fluorometers, radioactivity counters, colorimeters or chemiluminescence measuring devices.

In another preferred feature of the first embodiment, in step c), the affinity medium is a solid phase. These are, for example, microplate wells or beads, which are coated with an affinity ligand suitable for capturing affinity tags. More preferably, the microplate well is coated with a receptor selected from the group consisting of Ni ²⁺ , anti-tag antibody, glutathione or streptavidin. Most preferably, the microtiter well is coated with Ni ²⁺ and preferably the affinity tag is a histidine tag. Appropriate combinations of affinity tags and affinity ligands will be understood by those skilled in the art.
In another preferred feature of the first embodiment, step d) further comprises a washing step to remove unbound material, wherein the amount of label bound to the affinity medium is measured (or reconstituted) in an appropriate manner. RT polymerase activity is measured), in which case the substance bound to the affinity medium is proportional to the amount of labeled p51 / p66RT heterodimer.
As will be readily appreciated by those skilled in the art, the series of steps in the assay resulting in the incubation of p66 monomer and p51 can be modified to achieve the same result. For example, the p51RT subunit can be immobilized on a solid phase prior to incubation with the p66 subunit. Similarly, buffer dilutions that promote subunit association may be performed simultaneously with mixing of the p66 and p51 subunits, or after contacting the p66 solution with p51.
Similarly, contacting the test compound may be performed in a separate step without affecting the principles of the assay. For example, the test compound can be added to the p66 homodimer solution before addition of the dissociator; before mixing with the p51 subunit; before dilution with the reassociation buffer; or simultaneously with dilution with the reassociation buffer.

Example
Materials and methods
1. The gene of the 51 kDa subunit of the RT expression plasmid HIV-1RT (HXB2 strain) was cloned between the XhoI and HindIII restriction sites of the pBADHisB prokaryotic expression system (Invitrogen) to obtain pBAD-HisRT51. This construct allows expression of the RTp51 subunit as an N-terminal polypeptide histidine (6xHis) fusion protein following transformation with an appropriate bacterial strain (eg, JM109 strain of E. coli) with arabinose.
Site-directed mutagenesis using the Quick Change® position-directed mutagenesis kit (Stratagene) to provide specific labeling of the 66 kDa RT subunit containing reagents that allow fluorescence detection Induction was performed to produce P2C / C38S / C280SRT. In the above, two cysteine residues (C38, C280) of wild-type (WT) RT are removed, and one Cys residue is inserted near the N-terminus of the subunit.
The HIV-1RT wild-type and P2C / C38S / C280SRT (SEQ ID NO: 1) 66 kDa subunit genes were cloned between EcoR1 and HindIII restriction sites of pKK223-3 prokaryotic expression system (Amersham Pharmacia Biotech), respectively. -RT66WT and pKK-RT66-P2C were obtained. These vectors provide IPTG-dependent expression of the HIS-tagged 66 kDa RT subunit.

2. Reagents Delfia Eu-N1-iodoacetamide chelate and Eu ⁺³ standard, Delphia enriched solution and Delphia wash concentrate for TRF assay of RT dimer formation were purchased from Perkin Elmer.
Ultrapurified urea, tris (oxymethyl) aminomethane and water were obtained from Fluka. Magnesium chloride (SigmaUltra) and diethylenetriamine-tetraacetic acid (DTPA) were obtained from Sigma.
96-well Ni-NTA HisSorb plates (colorless) were purchased from Qiagen or Pierce. Fluorescence measurements were performed on a SpectraMAX Gemini XS microplate fluorometer (Molecular Devices).
3. Expression and purification of RTp51 subunit monomer and RTp66 / p66 homodimer
E. coli JM109 transformed with pBAD-HisRT51 was grown in “Terrific broth” to the middle logarithmic growth phase (OD ₆₀₀ was 0.5). Subsequently, p51RT expression was induced by the addition of 1% arabinose. After further incubation for 3 hours, the cells were collected by centrifugation and lysed using BugBuster® protein extraction reagent (Novagen). P51RT was purified from the lysate by Ni ^{+ 2-} NTA affinity chromatography using His-Bind resin (Novagen). Both lysis and extraction were performed according to the manufacturer's instructions. After elution from Ni ⁺² -NTA resin, it was passed through a NAP® 25 column (Amersham Pharmacia Biotech) twice and 50 mM Tris-HCl (pH 7.5, 4 ° C.) containing 60 mM magnesium chloride and 50% glycerol. P51RT buffer was replaced, and the final protein sample was stored as a partial preparation at −80 ° C. Purified p51RT exists exclusively as a monomer.
Expression of wild-type and mutant p66RT was induced by adding 1 mM IPTG to 1 liter of appropriately transformed mid-log phase E. coli JM109 cells (growth at 37 ° C. in “Terrific Broth”). . Bacterial growth was continued for an additional 5 hours at 32 ° C. after the addition of IPTG. Cells were harvested by centrifugation. Purification of p66RT was performed as previously reported (Fletcher et al. 1996, “Single step purification of HIV-1 recombinant wild type and mutant reverse transcriptase”, Protein Expression & Purification 7: 27-32). Purified p66RT exists exclusively as a homodimer.

4). Alkylation of P2C / C38S / C280S with p66 / p66RT Eu ^{+ 3-} N1- iodoacetamide chelate P2C / C38S / C280S p66 / p66RT was purified with 100 mM NaCl and 1 mM TCEP-hydrochloric acid (Tris (2-carboxyl The mixture was concentrated at room temperature to about 20 μM (about 2.5 mg / mL) in 50 mM Tris-HCl buffer (pH 7.9, 20 ° C.) containing (ethyl phosphine hydrochloride (Pierce)). A partial sample of Eu-N1-iodoacetamide chelate protein-modifying reagent (prepared as a 5 mM stock solution in DMSO) was added to give an approximately 20-fold molar excess over the p66 subunit concentration. The alkylation reaction was allowed to proceed for 6 hours at room temperature. Alternatively, the reaction may be performed overnight at 4 ° C. At the end of the reaction, excess DTT was added to consume unreacted Eu-N1-iodoacetamide chelate. The alkylated p66 / p66RT is passed through DTT- through two passes through a NAP® 25 column equilibrated with 25 mM Tris-HCl buffer (pH 7.5, 20 ° C.) containing 60 mM magnesium chloride and 50% glycerol. Separated from reagent conjugate. For label stoichiometry, it was found that Eu-N1-iodoacetamide was generally 0.7-1 mole per mole of p66RT subunit (ie, 1.5-2 mole / mole p66 / p66RT homodimer).

The kinetics characteristics of the mutant and chemically modified RT proteins indicate that p66 / p66RT of P2C / C38S / C280S has an RNA-dependent DNA polymerase (RDDP) specific activity comparable to the wild-type p66 / p66 homodimer enzyme. (Data not shown). P2C / C38S / C280S p66 / p66RT chemically alkylated with Eu-N1-iodoacetamide chelate resin retains about 80% of the RDDP activity of the unmodified protein (Table 1). Furthermore, the modified RT is equally sensitive to inhibition by TSAOe ³ T (Table 2) as the unmodified P2C / C38S / C280Sp66 / p66RT mutant (Table 2), and thus has the same sensitivity as wild-type p66 / p66RT ( Data not shown). Thus, the genetic and chemical processing of RT required for the preparation of reagents for dimerization assays appears to have little effect on enzyme reactions within the parameters of the assay protocol.

Table 1: Specific activity of RNA dependent DNA polymerase (RDDP) and DNA dependent DNA polymerase (DDDP) of P2C / C38S / C280Sp66 / p66RT

Table 2: Inhibition of P2C / C38S / C280Sp66 / p66RT by HIV RT inhibitors

5. Microplate assay to monitor p66-p51RT dimer formation The principle of this assay is the mixing of His6X-p51 and fluorophore labeled p66RT in solution, followed by His6X-p51 / fluorophore labeled p66RT heterodimer produced. Includes solid phase capture on Ni ²⁺ -NTA microplates. After washing to remove unbound material, the amount of fluorescence bound to the microplate is measured with a suitable fluorescence plate reader. Alternatively, the polymerase activity of the reconstituted RT can be determined using a fluorescent RNA / DNA intercalator (eg, PicoGreen®).
When purified recombinant p51RT is present as a monomer, purified p66RT is exclusively a dimer. Since the subunit association energy of the p66 / p66RT homodimer is only slightly less than that of the p51 / p66RT heterodimer, heterodimer formation after simple mixing of p51 and p66 / p66RT is very slow, and in high-throughput assays Inconvenient. Thus, a search was performed to determine the optimal type and concentration of denaturant needed to dissociate the p66 / p66 homodimer without significantly affecting the secondary structure of the protein. It has been found that urea is superior to guanidine chloride as a denaturing agent. The effect of urea concentration on p66 / p66 dissociation and protein denaturation (changes in protein secondary structure) is shown in FIG. The optimal urea concentration for maximizing the TRF signal in this dimerization assay system is presented in FIG.

Various data, including the data shown in FIGS. 1, 2 and 3, have shown that 1-3.5M urea is optimal. There are a number of tryptophan residues at the HIV-1RT subunit interface. They are essentially “buried” and therefore emit fluorescence of about 335 nm when RT is excited at a wavelength between 280-295 nm. As shown in FIG. 1A, these residues become “solvent exposed” as the urea concentration increases, as shown by the λ _max ^em red shift toward 350 nm. Become. At the same time, however, this urea concentration has little effect on the ellipticity of the RT molecule (FIG. 1B), suggesting that the secondary structure of the protein will not change significantly (ie, significant “denaturation” of the protein). "Did not occur). This suggests that increased solvent exposure of the tryptophan residue of the protein results primarily from dissociation of the RT subunit. The change in ANS binding to RT as a function of urea concentration (FIG. 1C) is consistent with this interpretation.
FIG. 2 shows the change in dimer formation of p66 / p66RT as a function of urea as determined by size exclusion chromatography. In this case, the homodimer dissociation appears to be essentially complete with 2M urea and the progression of denaturation / aggregation of the p66 monomer occurs when the peak slowly shifts to a higher molecular weight as the urea concentration increases. Seems to be. FIG. 3 shows that the labeled RTp66 / p66 homodimer and His-tagged p51 monomer were incubated at a urea concentration of between 0-6M, after which the time resolved RT sub-time was added to the nickel-coated microplate. Signals obtained in unit reassociation assay are shown. Therefore, 1-3.5 M urea was selected as the concentration range that appeared to provide the highest level of reassociation with minimal adverse effects on RT secondary structure.

Yet another experiment showed that using 50 mM Na ₂ SO ₄ over 100 mM MgCl ₂ in the regeneration buffer significantly enhanced the TRF-specific signal over the assay background. (Table 3).

Table 3: RT dimer formation assay as a function of regeneration buffer salt concentration
Signal to noise ratio

6). Antiviral activity of RT subunit disrupting substances and RT subunit association inhibitors
Assay Protocol The assay concept is shown in FIG. 5A. For a total volume of 20 μL, 100 pmol purified His6x-p51RT subunit and 200 pmol Eu-N1-iodoacetamide chelate labeled p66RT subunit (ie 100 pmol RTp66 / p66 homodimer) were urea (25 mM Tris-HCl (pH 7.5, Prepared at 20 ° C., containing 100 mM MgCl ₂ ) to provide a final concentration of 3.5 M urea. The mixture is incubated for 1 hour at room temperature.
Dilute this solution with 200 μL of the compound solution in question in a well of a 96-well Ni-NTA HiSorb plate (prepared with Tris-HCl buffer (25 mM, pH 7.5, 20 ° C.) containing 50 mM Na ₂ SO ₄ ) To do.
The dilution step provides a 10-fold dilution, reducing the urea concentration from 3.5M to 0.35M, thereby allowing the formation of RTp66 / p51 heterodimer. Dimerization is allowed to proceed for 6 hours, and the plates are subsequently washed 3 times with Delfia wash buffer containing 200 μM DTPA. After washing, 200 μL of Delphia enhancement solution is added to each well followed by 30 minutes incubation at room temperature. Fluorescence measurements were performed on a Molecular Devices SpectraMAX Gemini XS instrument using excitation, illumination and cutoff wavelengths of 335 nm, 615 nm and 530 nm, respectively. Using this format, TSAOm ³ T reduces the signal in this assay (FIG. 5B). TSAOm ³ T is a part of TSAOs and is thought to inhibit RT dimer formation to the same extent as TSAOe ³ T. In addition, nevirapine and efavirenz are both non-nucleoside reverse transcriptase inhibitors and do not inhibit enzymes through RT dimer formation. Indeed, the two NNRT inhibitors are chemical enhancers of HIV-1RT dimer formation and have been reported to cause inhibition of polymerase activity due to deleterious structural changes (Tachedjian et al. 2001, PNAS, 7188- 7193). In this unique assay format, the% signal relative to the control shown in FIG. 5B supports the above hypothesis indicating that the assay is useful for identifying inhibitors / enhancements in the process of HIV RT dimer formation.

  Alternatively, the polymerase activity of the reconstituted p66 / p51 reverse transcriptase may be measured. In this manner, the necessary substances (template primer, nucleotides, magnesium chloride) present in the appropriate buffer (Tris containing DTT, GSH and Chap, pH 7.8) following the dimer formation step are added directly to the wells, Incubate for an additional hour, after which the amount of polymerization reaction is measured by adding a fluorescent RNA / DNA intercalator. An example is as follows: homodimer p66 / p66 (10-20 pmol) of His-p51 (25-50 pmol) in 1 M urea (prepared with 20 mM Tris-HCl (pH 8.0) containing 250 mM sodium chloride). Incubate in the final volume of 10 μL for 1 hour in the presence to dissociate the homodimer. Subsequently, the first 5-fold dilution with reassociation buffer (50 mM Tris-acetic acid (pH 8.0), 500 mM sodium chloride, 0.05% Tween-20, 0.01% BSA) with test compound in association buffer for 1-2 hours. Perform and form heterodimers by reducing the urea concentration to 200 mM. The final step consists of capturing the reconstituted His-tagged heterodimer by transferring the sample to a well of a nickel plate containing 100 μL of reassociation buffer and incubation for 1 hour. The reaction is allowed to proceed for 1-2 hours at 37 ° C. after 3 washes, after which a polymerase assay cocktail is added and the extended RNA / DNA product is detected by a fluorescent intercalator, PicoGreen® .

7). Another assay format The biologically relevant and active form of HIV RT found in infectious virions is a heterodimer comprising two polypeptides (p66 and p51). p51 is derived from p66 by proteolytic cleavage of its C-terminal domain during viral maturation. The two subunits of the 66 kDa and 51 kDa forms are present in a 1: 1 ratio. This heterodimer RT is generated in a two-step dimer formation process. The kinetics involves a process in which the p66 and p51 subunits rapidly associate to form immature dimers, followed by a slow structural change that produces a fully active form (p66 + p51-> immature p66 / p51-> active p66 / p51 (Divita et al. 1995, J. Mol. Biol. 245: 508-521, 12, 13)).
However, more recent evidence has suggested another RT generation mechanism. The mechanism first involves the formation of a p66 / p66 homodimer from the p66 subunit, followed by the formation of a p66 / p51 heterodimer by HIV protease cleavage (Oral Presentation, Retrovirus 2001 Cold Spring Harbor, NY, May 26, 2001). It is unclear at this point which mechanism is dominant during viral replication, and screening assays designed to examine each pathway individually could provide new therapeutic approaches. Accordingly, it is another feature of the present invention to search for compounds with the potential to inhibit homodimer formation of the p66 subunit.

FIG. 1 shows the effect of urea concentration on HIV-1 p66 / p66RT protein structure. Panel A shows the change in maximum wavelength of intrinsic protein fluorescence emission as a function of urea concentration. An increase in the F350 / 335 ratio indicates increased exposure of the normally buried tryptophan residue to aqueous solvent. The increase in the ellipticity of the molecule at 222 nm is the magnitude of the disappearance of the RT secondary structure. Panel C shows the change in binding between 8-anilino-1-naphthalene-sulfonic acid (ANS) and RT. The change corresponds to the integrity of the RT dimer subunit. FIG. 2 shows the dimerization state of the p66 subunit as a function of urea concentration, as determined by size exclusion chromatography. FIG. 3 shows the optimal urea concentration used in the TRF (Time Resolved Fluorescence) assay for HIV-1 RTp66 / p51 heterodimer formation. FIG. 4 shows the time as a function of time after dilution of the urea-modified His6X-p51RT subunit and Eu-N1-iodoacetamide chelate labeled p66RT subunit in a “subunit association” buffer containing 100 mM MgCl _2. The TRF signal is shown. Details are described in the examples. FIG. 5 shows the inhibition of HIV-1RT dimer formation (panel B) by TSAO-m ³ T and other NNRTIs as measured by assay concept (panel A) and TRF.

Claims (27)

A method for measuring heterodimer formation of the p66 / p51 subunit of HIV RT comprising:
a) a first containing a p66 subunit homodimer in the presence of urea as a dissociator at a concentration of 1 to 3.5 M sufficient to dissociate the p66 subunit homodimer without affecting the secondary structure of the protein. Providing a solution;
b) Reassociation buffer (50 mM Na ₂₎ free of dissociator in an amount that brings the first solution into contact with the p51RT subunit and further reduces the dissociator concentration to one-tenth of the original concentration. Incubating in the presence of 25 mM Tris-HCl (pH 7.5, 20 ° C.) containing SO ₄ to allow the assembly of the reconstituted RT complex of the p66 / p51 RT subunit, wherein one of the subunits Comprises an affinity tag and the other of the subunits comprises a detectable label;
c) contacting the incubate of step b) with the affinity medium under conditions that allow the p66 / p51 complex to bind to the affinity medium; and d) washing to not bind to the affinity medium. Determining the amount of complex formed by measuring the level of detectable label bound to the affinity medium after removing the substance.   2. The method of claim 1, wherein in step d) the amount of the complex is determined by measuring the polymerase activity of the reconstituted RT complex. The urea is a concentration of 3 .5M, The method of claim 1.   2. The method of claim 1, wherein p51 of step b) has an affinity tag attached or has a detectable label. The method according to claim 4 , wherein an affinity tag is added to p51 of step b).   The method according to claim 1, wherein p66 of step b) has a detectable label or has an affinity tag attached thereto. 7. The method of claim 6 , wherein p66 of step b) has a detectable label. The method according to claim 4 or 6 , wherein the affinity tag of step b) is selected from the group consisting of a hexahistidine tag, FLAG, T7, HA, GST and biotin. 9. The method of claim 8 , wherein the affinity tag is hexahistidine. Wherein the detectable label is a fluorescent label, a radioactive atom, a chemiluminescent label, is selected from the group consisting of specific detectable antibodies, and enzyme markers tag bound to p66-specific or p66, claim 4 or 6 The method described in 1. 11. The method of claim 10 , wherein the detectable label is a fluorescent label selected from the group consisting of fluorescein, Oregon green, rhodamine, Texas red, phycoerythrin, and Eu3 ⁺ . The method of claim 11 , wherein the fluorescent label is Eu ³⁺ . The detectable label of step b) is added to N- end terminal stain residues p6 6 consisting of the amino acid sequence shown in SEQ ID NO: 1, The method of claim 1.   The method of claim 1, wherein the affinity medium of step c) captures any reconstituted labeled p66 / tagged p51 / RT heterodimer.   The method according to claim 1, wherein in step c) the affinity medium is a solid phase coated with a receptor suitable for capture of the affinity tag. The method of claim 15 , wherein the solid phase is coated with a receptor selected from the group consisting of Ni ²⁺ , anti-tag antibody, glutathione and streptavidin. The method of claim 16 , wherein the solid phase is coated with Ni ²⁺ . The method of claim 15 , wherein the affinity tag is histidine. The method of claim 15 , wherein the solid phase is a microplate.   Step d) further comprises a washing step for the purpose of removing unbound material, measuring the amount of affinity medium bound label in an appropriate manner (or measuring the polymerase activity of the reconstituted RT) and binding to said affinity medium. 2. The method of claim 1, wherein the material obtained is proportional to the amount of labeled p51 / p66RT heterodimer. A method for identifying a compound capable of modulating HIV RT heterodimer formation comprising:
Performing steps a) to d) according to claim 1 in the presence or absence of a test compound, and e) comparing a sample of said test compound with a control sample lacking said test compound, Wherein the modulation of p66 / p51 complex formation of the test compound sample comprises the step of showing the ability of the compound to modulate heterodimer formation. A method of identifying a compound capable of interfering with HIV RT heterodimer formation comprising:
Performing steps a) to d) according to claim 1 in the presence or absence of a test compound, and e) comparing said test compound sample with a control sample lacking said test compound, thereby A method wherein the reduction of p66 / p51 complex formation of a sample of the test compound comprises the ability of said compound to inhibit heterodimer formation. A method for identifying a compound capable of promoting HIV RT heterodimer formation comprising:
Performing steps a) to d) according to claim 1 in the presence or absence of a test compound, and e) comparing said test compound sample with a control sample lacking said test compound, thereby An increase in p66 / p51 complex formation of the test compound sample indicates the ability of the compound to promote heterodimer formation,
A method comprising the steps of: A method for measuring homodimer formation of the p66 subunit of HIV RT comprising:
a) providing a first solution comprising a first p66 subunit homodimer in the presence of urea as a dissociator at a concentration of 1 to 3.5 M ;
b) the said first solution and a second p66 subunits homodimers are contacted in the presence of said dissociation agent, in an amount to further reduce the concentration of the dissociating agent in one tenth of the original concentration, the dissociation Incubating in the presence of agent-free reassociation buffer ( 25 mM Tris-HCl, pH 7.5, 20 ° C. containing 50 mM Na ₂ SO ₄ ) to allow association of the p66 / p66RT subunit complex Wherein one of the subunits comprises an affinity tag and the other of the subunits comprises a detectable label;
c) contacting the incubate of step b) with an affinity medium under conditions that allow the p66 / p66 complex to bind to the affinity medium; and d) of the detectable label bound to the affinity medium. Determining the amount of complex produced by measuring the level (or by measuring the reconstituted RT polymerase activity). A method for identifying a compound capable of modulating HIV RT homodimer formation comprising:
Performing steps a) to d) according to claim 24 in the presence or absence of a test compound, and e) comparing said test compound sample with a control sample lacking said test compound, thereby Wherein the modulation of p66 / p66 complex formation of the test compound sample comprises the ability of said compound to modulate homodimer formation. A method for identifying a compound capable of interfering with HIV RT homodimer formation comprising:
Performing steps a) to d) according to claim 24 in the presence or absence of a test compound, and e) comparing said test compound sample with a control sample lacking said test compound, thereby A method wherein the reduction of p66 / p66 complex formation of a test compound sample comprises the ability of said compound to inhibit homodimer formation. A method for identifying a compound capable of promoting HIV RT homodimer formation comprising:
Performing steps a) to d) according to claim 24 in the presence or absence of a test compound, and e) comparing said test compound sample with a control sample lacking said test compound, thereby The method wherein the increase in p66 / p66 complex formation of the test compound sample comprises the ability of said compound to promote homodimer formation.

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